This Competitive Revision Application for 4R00DE018188-02 proposes to expand the parent project to include a non-viral method of gene delivery based upon ultrasound destruction of DNA-associated microbubbles. This ultrasound-assisted gene transfer (UAGT) works differently from the viral-based methods of the parent project, but the end result, gene transfer to the salivary gland is the same. Therefore, this supplement is proposed as a parallel but equivalent track, bringing in a co-investigator from within the Pi's affiliated institution who has almost twenty years of experience with ultrasoundmicrobubble technology. Figure 1 illustrates the relationship of the parent project and proposed supplement. By executing the parent project and the proposed supplemental research plan in concert, we will be able to leverage the existing expertise acquired in the physiological and molecular analysis of salivary gland-based gene therapy for Fabry disease. This will create a climate where the translational potential of both viral and non-viral gene transfer methods can be objectively compared under tightly controlled conditions. With salivary gland-based gene therapy now in a Phase 1 human clinical trial, such comparisons are imperative if long-term gene expression in the salivary gland of humans is to be achieved. The major mechanism for cementing collaboration between the Pl and the Co-l is the support of a floating graduate student who will participate in every step of the proposed supplemental project. This mechanism will link Dr. Wheatley's expertise in the construction and destruction of ultrasound microbubble contrast agents with Dr. Passineau's gene therapy laboratory. The proposed budget includes allocation for this graduate student, based in the Drexel University School of Biomedical Engineering, to make frequent extended visits to Pittsburgh for animal work and sample analysis.